Cell lines and cell culture
The human HCC cell line HepG2 and Huh7 were obtained from the Chinese Academy of Sciences Cell Bank (Shanghai, China). The cells were cultured in the DMEM/ High Glucose (hyclone, USA) in a humidified incubator at 37 °C temperature and 5% CO2 concentration. 10% FBS (Fetal bovine serum, Gibco USA) and Penicillin-Streptomycin (100 U/mL and 100μg/mL respectively) supplementations were added in the DMEM/ High Glucose medium prior to culture.
Constructions of Plasmid and cell transfection
SNHG5 and UPF1 overexpression plasmids, SNHG5 knockdown plasmids (SNHG5 shRNA with a corresponding negative control shRNA-NC), SNHG5-Mut/WT plasmids (pCMV-SNHG5- Mut vector containing mutations at the putative UPF1 binding site were generated by site-directed mutagenesis) and the siRNA (small interfering RNA) against UPF1, were designed by Genepharma ((Shanghai, China). Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) was used to transfect the HCC cells with plasmids by the manufacture’s protocol. The stable clones were selected by 5μg/ml Puromycin contain medium. The Puromycin-resistant cell clones were established after 4 weeks. Gene expression level was evaluated by Quantitative real-time PCR.
Cell proliferation assays
MTT (3-[4, 5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay MTT(0.5 mg/ml) added into transfected cells and keeping in the dark for 4 hours. Then, removed the supernatant, added 150μl DMSO and measured the optical density (OD) at 490 nm.
EdU incorporation assay Transfected cells were seeded in to a 96 well plate (2×103) with complete growth meium. Then according to the manufactures protocol with the EdU detection kits (Keygen, Nanjing, China) the process was carried out. The experiments were done in triplets.
Colony formation assay After routine incubation, transfected cells were trypsinized, centrifuged, counted and replated at a density of 500 cells/6 cm plate. After 12 days, the cell colonies (one colony containing at least 50 cells) were fixed with 3.7% methanol, stained with 0.1% crystal violet and counted.
Ultra-low attachment culture dishes (Corning, USA) were used to culture HepG2 and Huh7 cells with DMEM/F12 (Gibco, USA) added with 1% FBS, 20 ng/mL epithelial growth factor, and 20 ng/mL fibroblast growth factor for two weeks. The formation and the number of spheroids were detected by a stereomicroscope (Olympus, Japan).
RNA isolation and quantitative real-time PCR
The total RNA from the cultured cells and collected HCC tissues were extracted form Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to manufacture’s protocol. The Prime Script TM RT Master Mix Kit (Takara, Japan) and Mir-X miRNA qRT-PCR SYBR Kit (Takara, Japan) were used to obtain the cDNA. Quantitative real-time PCR (qRT-PCR) was performed with SYBR Premix Ex Taq™ II (Takara)on Thermal Cycler CFX6 System (Bio- Rad). β-actin as the endogenous controls of qRT-PCR. The 2−ΔΔCt method was used o calculate relative gene expression. Primers sequences for PCR were presented in Supplementary Table1.
Western blot analysis
The total protein from the cultured HCC cells and the tissue samples were isolated by RIPA (Beyotime, Haimen China) supplemented with proteinase and phosphatase. BCA detection kit (Keygen, Nanjing, China) was used for qualification according to the manufacture’s protocol. For electrophoresis 5% gel was used for concentration and 10% for separation. Following electrophoresis the proteins were transferred on a PVDF membrane (Merck Milipore) and were blocked by 5% non-fat milk for 1 hour. Then the PVDF membrane was incubated overnight at 40C with the primary antibodies (Supplementary Table 2). On the next day the secondary antibody (Zhuangzhi Biology, China) was diluted 1:5000 ratio in TBST, and the membrane was re-incubated for 1 hour. The protein bands were evaluated by ECL immunoblotting kit following the manufacture’s protocol ( Milipore, USA).
Cells were fixed in 4% paraformaldehyde at room temperature for 15 min after culturing on glass coverslips for 24 h. Then washed by PBS. The adherent cells were permeabilized using 0.5% Triton X-100, blocked with 10% goat serum in for 1h. Then, incubated with primary antibody 4 °C overnight and secondary antibodies with an appropriate dilution. After washing three times with PBS, coverslips were stained with DAPI and imaged with a invert fluorescent microscope (Nikon Eclipse Ti-S).
Tumor formation in BALB/c nude mice
4 weeks aged BALB/c nude mice were randomly divided into two groups, which were purchased from the Central Laboratory of Animal Science, Xi’an Jiaotong University, China. The mice were kept under Sterile Specific-Pathogen free (SPF) environment. Then, 5 × 106/200μl HepG2 cells stably transfected with SNHG5-shRNA or NC-shRNA were subcutaneously injected. The tumor formations were observed every 4 days interval. After 5 weeks injecting, mice were sacrificed. The study was done according to the Guide line for the “Care and Use of Laboratory Animals of the National Institutes of Health” and was approved by the Medical Ethics Committee of the Experimental Animal Center of Xi'an Jiaotong University.
Luciferase reporter assay
Before transfection the HCC cells were placed and cultured in a 96 well plate til 60-80% proliferation was achieved. Wild and mutant reporter plasmids of SNHG5 (SNHG5-WT-luc, SNHG5-MuT-luc) were co-transfected with UPF1 plasmid respectively. The alteration in the luciferase activity was devaluated in each group by Dual Luciferase Assay Kit and as a control Renilla luciferase activity was employed.
Millipore EZ‐Magna RIP RNA Binding Protein Immunoprecipitation kit (Millipore) was applied to performed RIP assays according to the manufacturer's protocol. Rabbit polyclonal IgG (Millipore) and antibodies to UPF1 (Abcam) were applied in RIP assays. Then, RIP‐PCR was performed, and total RNA was used as input controls.
SPSS 23.0 (IBM, SPSS, Chicago, IL, USA) and GraphPad Prism V7.0 (GraphPad Software, CA, USA) were used to analyzed the results. Student’s t-test was done to evaluate the difference between two groups. A two-tailed P< 0.05 was considered as statistically significant and P < 0.01 was very significant.